In order to understand the development and functioning of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created a RT-PCR based subtracted cDNA library from fetal thymic stromal cells. The novel cDNAs were then screened for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Multiple full length cDNA clones were isolated from a SCID thymus library. The three novel genes were named Epithin, Spatial, and Thymic Stroma Co-Transporter (TSCOT). We are currently focusing on experiments with two of these gene products (TSCOT and Epithin). 1). The lab has been making good progress in generating model mouse systems which show a thymus-specific expression pattern of targeting of exogenous genes specifically in different thymic epithelial cell compartments using both transgenic and knock-in mouse approaches. A transgenic mouse line containing 3.1kb of the TSCOT promoter driving Enhanced Green Fluorescent Protein (EGFP) was established to identify the thymic stromal cell lineage in which TSCOT is expressed. Surprisingly, this mouse showed EGFP expression in the subcapsular and medullary compartments of the thymus in contrast to the cortex where the endogenous gene is expressed. In another model, a mouse was generated by knocking in the LacZ gene at the TSCOT locus. We have shown that LacZ is expressed only in the thymic epithelial cell compartment. Interestingly, expression of these targeted proteins changes during development. Using this information, we are now generating more mouse lines that can be used for specific cell ablation in either a constitutive or inducible fashion in different thymic stromal cell compartments. 1)During the last year, using our established mouse models, in addition to previously available mutant mice, we investigated the process of thymic epithelial cell development with a focus on cortical / medullary compartmentalization and differentiation. In addition, using a targeted neoself protein in thymic epithelial cells, we tested whether self tolerance could be established by an antigen expressed in thymic epithelium. 2) We were successful in detecting message for CRE in a newly generated transgenic line harboring both the CRE and EFGP genes under the control of the TSCOT promoter. 3). In Collaboration with Dr. Colin Anderson, we tested the role of thymic cortical epithelium in the induction of self tolerance. Previously it was suggested that cortical epithelium can only positively select thymocytes but not negatively select them. However, using thymic transplantation under the kidney capsule, we have found that T cells can accept a thymus graft expressing their own kind of MHC II molecules only in the cortical epithelium, indicating that antigens present in the cortical epithelium can induce self tolerance. 4). In collaboraion with Dr. D. Park, we have been successful in identifying proteins involved in interactions with Epithin using Yeast 2 hybrid technology. In addition, we are continuing our search for the general nature of Epithin's function and the mechanism of its processing during cellular signaling. One of the proteins isolated is an actin-binding protein Filamin, which in fact behaves as a molecular bridge linking the extracellular membrane protein Epithin to the key intracellular cytoskeletal protein actin. When Filamin links Epithin into cortical actin in the cell, Epithin is concentrated in the cell contact sites and a protease activity is then transactivated, resulting in the shedding of the short form of the Epithin protein.